Divide and rule: localised Ca2+ signalling in sensory neurons

Lead Research Organisation: University of Leeds
Department Name: Sch of Biomedical Sciences

Abstract

In order to perceive and evaluate the environment, animals and humans are equipped with a special part of the nervous system, the somatosensory system. It includes nerves that run through our body and collect information about surrounding conditions (such as temperature and stiffness of objects we touch) and also about our own body's integrity. These nerves contain various molecular sensors that respond to specific external events (like touching a hot object) by producing miniature electrical signals that are then sent to the brain for interpretation. A single nerve usually possesses a range of different sensors to respond to different stimuli, yet the electrical signals produced by different stimuli are very similar. A major puzzle in the field of sensory physiology is how different types of signals are specifically interpreted by a single sensory nerve cell; the overarching aim of this proposal is to attempt to solve this puzzle.

Based on the wealth of our preliminary data and published work from our group and others, we propose the idea that may explain such signal coding: different signalling mechanisms might be 'packed' in specific spots separated in space at certain areas within the nerve cell. Such physical separation of one signalling machinery from another allows nerve cells to use common signalling events and messenger molecules without 'mixing up' the meaning of the message.

We are focusing on one specific aspect: how a nerve responds to local inflammation, a process that often results in pain (such as toothache or arthritic pain) and how these responses are different form nerve's responses to other sensory stimuli.

Our group's work points to one specific structure formed at junctions between the outer barrier ('plasma membrane') of a nerve cell and a cistern-like organelle called the 'endoplasmic reticulum', which is found in the nerve cell itself. Such junctions form tiny spaces ('nanodomains') which are relatively well separated from the rest of the cell interior and contain a slew of sensors responsible for detection of tissue inflammation. Once these sensors are triggered, the cellular machinery activates the sensory nerve cell and ultimately results in the feeling of inflammatory pain. We have already identified some components of this junctional structure and its overall importance for inflammatory pain. However, hardly anything is known about how these junctions are formed and maintained, how the key proteins within the junction are arranged and how these change in response to inflammation. Our project aims to close this gap in our knowledge with an ultimate goal of learning how to disturb this pathway and separate inflammation from pain. This proposal has three specific aims:

1) Identify key components responsible for communication in the sensory nerve's junctional space.
2) Discover how these components interact when a nerve cell responds to inflammation.
3) Develop techniques to study these junctional spaces at the tips of nerve endings.

We have developed a well-rounded and multidisciplinary approach combining cutting-edge methods such as super-resolution microscopy, proteomics, molecular and cell biology approaches and animal behavioural studies. We are confident that this research will bring our understanding of nerve cell signal coding to a new level of insight. Specifically, we will unravel complex molecular machinery responsible for production of inflammatory pain sensation. This new knowledge may shape future methods for pain treatment.

Technical Summary

This proposal focuses on intricate molecular mechanisms underlying inflammatory pain. In pain-sensing (nociceptive) sensory neurons, junctions between the plasma membrane (PM) and endoplasmic reticulum (ER) form signalling nanodomains harbouring molecular machinery for generating local Ca2+ signals. These signals excite nociceptors leading to generation of inflammatory pain. We have identified several key molecules involved in such Ca2+ signaling, including excitatory ion channels ANO1 and TRPV1, pro-inflammatory G-protein coupled receptors, inositol 1,4,5-trisphosphate receptors, as well as molecular components of Ca2+ release activated Ca2+ channel, CRAC. We also identified a scaffolding protein, junctophilin-4, as a crucial molecule necessary for junctional signalling integrity. These findings are revealing, yet the molecular architecture and dynamics of the junctional nanodomains and resident signalling complexes are only beginning to emerge. The goal of this proposal is to elucidate, on a nanoscale level, the junctional nanodomain machinery enabling nociceptors to respond to the inflammatory environment and generate the nociceptive input in a reliable, stimulus-specific manner. We have assembled a versatile toolkit, including single-molecule localization approaches (DNA-PAINT, proximity ligation), live imaging, cell and molecular biology approaches and in vivo tests, for comprehensive interrogation of junctional signalling in sensory neurons, covering molecular, cellular and whole organism levels. We will use this knowledge base and methodology to elucidate the role of junctional scaffold proteins in localised Ca2+ signalling in sensory neurons and reveal the dynamics of junctional nanodomains during the inflammatory activation. Finally, we will investigate junctional Ca2+ signalling in situ, at the nerve terminals. The outcome of this research will reveal key principles of intracellular signal transduction and may offer new insights for pain therapeutics.

Publications

10 25 50
 
Description The project focuses on how nerve cells respond to local inflammation, a process that often results in pain, and how these responses are different form nerve's responses to other sensory stimuli. We investigate specific structures formed at junctions between the outer barrier ('plasma membrane') of a nerve cell and a cistern-like organelle called the 'endoplasmic reticulum', which is found in the nerve cell itself. Such junctions form tiny compartments ('nanodomains') which are relatively well separated from the rest of the cell interior and contain a complex molecular machinery responsible for detection of tissue inflammation. Our project aims to unpick inner clockwork of this molecular machinery. Below are specific aims of the project with brief outline of the key findings.

Aim 1) Identify key components responsible for communication in the sensory nerve's junctional space.

Aim 2) Discover how these components interact when a nerve cell responds to inflammation.

We have identified novel molecular mechanism of amplification of inflammatory signals by sensory nerve cells via close interaction between two sensory ion channels, TRPV1 and ANO1; this work was published in the journal 'Science Signaling' in 2021. In addition, we discovered a role for the junctional protein called Extended synaptotagmin-1 in the dynamic interaction between the components of the neuronal signalling machinery in response to local inflammation. These findings were reported at the international meetings of the Biophysical Society (2023, 2024) and the Society for Neuroscience (2022) and are being prepared for publication. We also developed super-resolution microscopy methods to investigate interaction of proteins within neuronal nanodomains, these are published in journals 'Methods' (2021) and 'Cells' (2024).

Aim 3) Develop techniques to study these junctional spaces at the tips of nerve endings.

We have developed a number of new approaches to investigate signaling events at the tip of the nerve. One approach includes organoid-like culture of sensory neurons on a specific chips, called 'microfluidic chambers'. This approach allows realistic reconstitution of a single nerve in vitro in combination with unprecedented experimental access to all major compartments of the nerve, including nerve endings. The approach also helps to reduce the use of animals. This work is ongoing but the key technology is in place.

With this essential progress along all the specific aims, the project is well on track for completion.
Exploitation Route There are several ways the outcome of this research will be taken forward. First, the scientific discoveries made throughout the project may result in new ideas for pain treatment and management, for instance, the coupling between various proteins within junctional nanodomains can be targeted to 'decouple' inflammation form pain. Second, our findings reveal general principles of signal specificity and localization in the biological signaling, some of these will be applicable outside the pain and/or neuroscience research fields. Third, the super-resolution microscopy methods we developed and shared are applicable to a wide range of cellular and molecular studies, well beyond neuroscience and pain research. In fact, we developed these methods in a collaboration with cardiac physiology group.
Sectors Education

Healthcare

Pharmaceuticals and Medical Biotechnology

 
Title A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types 
Description A correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the Ca2+ channels in primary cells. This is achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT super-resolution imaging of the Ca2+ channels. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact Most super-resolution microscopy methods require fixed cells/tissue. Here we developed a correlative approach in which live signaling events recorded from the living cells can be matched to the nanoscale structural information obtained with super-resolution imaging. This will further the scope of super-resolution imaging. 
 
Title All-optical monitoring of chloride channel activity in living cells 
Description We developed a triple-wavelength fluorescent imaging approach to simultaneously perform halide-sensitive EYFP quenching (to measure Cl- channel activity) and ratiometric fura-2 Ca2+ imaging. The method can be used as a higher-throughput alternative to patch-clamp for studying Ca2+-activated Cl- channels. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact The publication is currently under revision. 
 
Title Correlative microscopy protocol to examine the nanoscale patterns of Ca2+ channels and individual Ca2+ sparks in sensory neurons 
Description We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of ryanodine and IP3 recetpors in the locality of each Ca2+ spark 
Type Of Material Technology assay or reagent 
Year Produced 2024 
Provided To Others? Yes  
Impact This method will allow scientists to gain single-molecule level insight in intracellular Ca2+ signaling events. 
URL https://www.mdpi.com/2073-4409/13/1/38
 
Title Super-resolution analysis of the origins of the elementary events of ER calcium release in dorsal root ganglion neurons 
Description This is the data supplement for the paper entitled, "Super-resolution analysis of the origins of the elementary events of ER calcium release in dorsal root ganglion neurons"There are two principal subdirectories within the enclosed zip file: 10xEExM_data: The directory contains two exemplar datasets each of 10x Enhanced expansion microscopy images of IP3R1 and RyR immunolabelling in DRG soma, at the sub-plasmalemmal regions.  Correlative Analysis: The directory contains two sub-directories of worked examples of data and correlative analysis of calcium sparks and dSTORM images of RyR and IP3R. The instructions for the code, run in IDL, are included in the Readme.txt enclosed within. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
URL https://zenodo.org/doi/10.5281/zenodo.10157969
 
Description Super-resolution imaging method development 
Organisation University of New South Wales
Country Australia 
Sector Academic/University 
PI Contribution We are providing tissue samples, primary neuronal culture methodology, genetic constructs, as well as time of a PDRA and a PhD student from our group.
Collaborator Contribution Supper-resolution method development expertise, imaging setup and software
Impact Super-Resolution Analysis of the Origins of the Elementary Events of ER Calcium Release in Dorsal Root Ganglion Neurons. Hurley ME, Shah SS, Sheard TMD, Kirton HM, Steele DS, Gamper N, Jayasinghe I. Cells. 2023 Dec 23;13(1):38. doi: 10.3390/cells13010038. A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types. Hurley ME, Sheard TMD, Norman R, Kirton HM, Shah SS, Pervolaraki E, Yang Z, Gamper N, White E, Steele D, Jayasinghe I. Methods. 2021 Sep;193:27-37. doi: 10.1016/j.ymeth.2020.10.005
Start Year 2020
 
Description Super-resolution imaging method development 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing tissue samples, primary neuronal culture methodology, genetic constructs, as well as time of a PDRA and a PhD student from our group.
Collaborator Contribution Supper-resolution method development expertise, imaging setup and software
Impact Super-Resolution Analysis of the Origins of the Elementary Events of ER Calcium Release in Dorsal Root Ganglion Neurons. Hurley ME, Shah SS, Sheard TMD, Kirton HM, Steele DS, Gamper N, Jayasinghe I. Cells. 2023 Dec 23;13(1):38. doi: 10.3390/cells13010038. A correlative super-resolution protocol to visualise structural underpinnings of fast second-messenger signalling in primary cell types. Hurley ME, Sheard TMD, Norman R, Kirton HM, Shah SS, Pervolaraki E, Yang Z, Gamper N, White E, Steele D, Jayasinghe I. Methods. 2021 Sep;193:27-37. doi: 10.1016/j.ymeth.2020.10.005
Start Year 2020
 
Description Supperesolution imaging 
Organisation University of Texas
Department Health Science Center at San Antonio
Country United States 
Sector Academic/University 
PI Contribution I am providing the tissue samples and consumables, as well as primary neuronal culture method.
Collaborator Contribution Time allocated at their NIKON STORM microscope; a postdoc in UTHSCSA actually performs the imaging. I have visited the collaborators in 2021 and 2024 and performed some new experiments, which are a part of NIH R01 grant application submitted for funding in November 2023.
Impact Local Ca2+ signals couple activation of TRPV1 and ANO1 sensory ion channels. Shah S, Carver CM, Mullen P, Milne S, Lukacs V, Shapiro MS, Gamper N. Sci Signal. 2020 Apr 28;13(629):eaaw7963. doi: 10.1126/scisignal.aaw7963.
Start Year 2014